Fluorescence methods are extremely widespread in chemistry and biology. The methods give useful information on structure, distance, orientation, complexation, and location for biomolecules [1]. In addition, time-resolved methods are increasingly used in measurements of dynamics and kinetics [2]. As a result, many strategies for fluorescence labeling of biomolecules, such as nucleic acids, have been developed [3]. In the case of DNA, one of the most convenient and useful methods for fluorescence labeling is to add a fluorescent moiety during the DNA synthesis itself. Addition of the fluorescent moiety during DNA synthesis avoids the extra steps required for post-synthesis labeling and purification. The majority of labels commonly used during DNA synthesis are attached to the DNA by tethers that are often 5 to 11 atoms long. These flexible tethers can at times be problematic, since they allow the dye to tumble independently of the DNA and -make the location of the dye difficult to determine precisely [4]. There are very few examples of dye conjugates that hold the dye close to the DNA, thus avoiding these problems. Among the known dyes of this class are ethenodeoxyadenosine [5] and 2-aminopurinedeoxyriboside [6]. These latter two compounds have modified DNA bases that are themselves fluorescent, and have found much use as probes of enzymatic activities such as DNA synthesis, editing, and repair [7-9].
The present invention provides fluorescent labels for nucleic acids which, rather than modifying an existing nucleic acid base, replace one or more DNA or RNA bases with a fluorescent cyclic compound. Since the replacement fluorescent cyclic compound is also a flat cyclic structure, only small perturbations to the overall nucleic acid structure occur upon its use. The fluorescent label may be thought of as a nucleoside analog in which a known fluorescent cyclic compound is joined to a carbon atom of a sugar moiety. The subject nucleoside analogs allow for close interaction, including stacking, with a neighboring RNA or DNA helix. There are many known cyclic fluorophores which may be joined to a carbon atom of a sugar moiety to form the nucleoside analogs of the present invention. Many of the known cyclic fluorophores have high quantum yields with varied excitation and emission characteristics. Moreover, their lack of functional groups makes them relatively simple to work with in preparing conjugates.
The literature has reported incorporation of 4-methylindole, naphthalene, phenanthrene, and pyrene fluorophores at the C1-position of deoxyribose [10,11]. In a similar strategy, the substitution of a coumarin dye at the C1 position of deoxyribose has also been reported[12]. The methylindole derivative has recently found use as a fluorescent reporter of DNA repair activities [13]. In addition, the C1xcex1 pyrene derivative has been shown to be useful in DNA diagnostics strategies, where it efficiently forms excimers with neighboring pyrene labels [14]. The C1xcex2 pyrene derivative stabilizes DNA helices markedly (due to its low polarity) [15,16], and can be enzymatically incorporated into the DNA helix [17]. Thus, this new nucleic acid labeling strategy has many useful applications.
The present invention provides nucleoside analogs with improved fluorescence characteristics, increasing the range of emission wavelengths over those previously studied. The subject nucleoside analogs are more generally useful in biophysical and diagnostics applications. These new compounds significantly broaden the range of fluorescence properties available for automated incorporation into DNA.
Although not all interactions between fluorophores are well understood, it is clear that there is more than one type of interaction between light-absorbing molecules. One useful class of interaction is Forster energy transfer, also called fluorescence resonance energy transfer, or FRET. In this interaction, fluorescence emission is transferred from a donor to an acceptor fluorophore. The extent of transfer depends on distance and on overlap in emission and absorption of donor and acceptor. FRET can occur over relatively long distances (tens of Angstroms). A second form of energy transfer is exciplex formation, which involves bonding between an excited-state fluorophore and a neighboring ground-state fluorophore. This results in a long red shift to fluorescence. Exciplexes can form only between molecules in direct contact or very nearly so. Exciplexes between two of the same molecules are known as excimers. Another class of interaction involving a fluorophore is quenching, in which a molecule causes the quantum yield of nearby fluorescent molecule to be lowered.
These forms of energy transfer are not well explored in systems where more than two chromophores are involved. FRET is now well known between pairs of well understood and widely used dyes, such as fluorescein, rhodamine, acridine, or cyanine dyes. Heretofore, FRET between more than two dyes has been unknown and unexplored. Similarly, while interactions between a few excimer-forming dyes such as stilbene and pyrene are known, exciplex interactions have not been widely explored for combinations of dyes. Little is known about the interactions among more than two fluorophores. Reasons for the dearth of study in this area include lack of available methods for assembling fluorophores in a regular designed fashion. The study of more complex molecules could entail an inordinate number of combinations even where only a few dyes are used. Assembling even a small fraction of these possibilities for study heretofore has been a daunting task. Even if carried out, many combinations of fluorophores lead to undesirable properties such as quenching. For example, placing two fluorescein labels close together results in very weak fluorescence emission.
The present invention allows for the generation of nucleoside analogs and nucleic acids incorporating the subject nucleoside analogs resulting in many types of fluorescence properties such as emission wavelength, emission intensity, and Stokes shift. Combinatorial arrays of fluorophores built on a nucleic acid backbone may be generated and screened for fluorophores having useful fluorescent properties such as high molar absorptivities which leads to high localized fluorescense intensities. Fluorophores providing multiple energy transfer, leading to very large Stokes shifts may also be identified from a library of combinatorial arrays. Large Stokes shifts are useful in avoiding background interference in fluorescence. FRET and exciplex forms of energy transfer usually lead to large changes in emission wavelength, resulting in many possible colors for ease of detection.
The present invention provides nucleoside analogs comprising a fluorescent cyclic compound joined to a carbon of a pentose, hexose, ribose or deoxyribose sugar moiety in either an xcex1 or xcex2 configuration. In a preferred embodiment, the fluorescent cyclic compound is joined to the C1 position of the sugar moiety.
Examples of fluorescent cyclic compounds which may be joined to the sugar moiety include oligomers of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). Preferably, an oligomer has a length of from 1 to 16. Terthiophene and sexithiophene are examples of oligothiophenes useful as cyclic compounds in the nucleoside analogs of the present invention.
Benzoterthiophene and terbenzothiophene are examples of oligobenzothiophenes useful as fluorescent cyclic compounds joined to a sugar moiety. Dimethylamino stilbene and styrylstilbene are examples of oligo(phenylene vinylenes) useful as fluorescent cyclic compounds joined to carbon of a sugar moiety.
Diphenylacetylene and phenyl (ethynyl) diphenylacetylene are examples of oligo(phenylene acetylenes) useful as fluorescent cyclic compounds joined to a carbon of sugar moiety.
Other fluorescent cyclic compounds useful as fluorescent cyclic compounds joined to a carbon of a sugar moiety include p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide.
Also provided are nucleoside analogs comprising a non-fluorescent cyclic compound joined to a carbon of a pentose, hexose, ribose or deoxyribose sugar moiety wherein the cyclic compound is cyclohexane, cyclohexene, decalin, benzene or dimethylamino benzene.
The nucleoside analogs of the present invention may be derivatized at an available carbon position with a substituent selected from the group consisting of methoxy, ethoxy, alkoxy, alkyl, dimethylamino, diethylamino, nitro, methyl, cyano, carboxy, fluoro, chloro, bromo, iodo and amino.
The present invention also provides nucleic acid molecules comprising at least one subject nucleoside analog. Oligomers comprising the subject nucleoside analogs are also provided.
Also in accordance with the present invention, there are provided phosphoramidite derivatives of the subject nucleoside analogs wherein the phosphoramidite is joined to the sugar moiety at the 3xe2x80x2 position. Examples of phosphoramidite derivatives include N,N-diisopropyl-O-cyanoethyl phosphoramidite or O-methyl-phosphoramidite derivatized at the 3xe2x80x2 alcohol of the nucleoside analog.
The present invention also provides nucleoside 5xe2x80x2-3xe2x80x2-paratoluoyl diesters derivatized at the C-1 atom of a sugar moiety with a fluorescent cyclic compound.
Methods of synthesizing the subject nucleoside analogs are also provided. The methods comprise the steps of coupling an organocadmium or organozinc derivative of a fluorescent cyclic compound to a carbon of Hoffer""s xcex1-chlorosugar and removing the protecting groups with a methanolic base.
Also provided are methods of synthesizing a phosphoramidite derivative of a subject nucleoside analog. The method comprises:coupling an organocadmium or organozinc derivative of a fluorescent cyclic compound to a carbon atom of Hoffer""s a-chlorosugar, removing the protecting groups with a methanolic base; tritilating the 5xe2x80x2-OH with dimeoxytritylchloride in the presence of a base; and phosphytilating the 3xe2x80x2-OH with a phosphytilating agent.
In addition, the present invention provides a method of preparing a fluorescently labeled nucleic acid molecule which comprises incorporating a subject nucleoside analog into an RNA or DNA molecule under conditions sufficient to incorporate said nucleoside.
A method of detecting a target nucleic acid in a sample to be tested is also provided. The method comprises contacting the target nucleic acid with a nucleic acid probe comprising at least one subject nucleoside analog for a time and under conditions sufficient to permit hybridization between the target and the probe and then detecting said hybridization.
Also provided by the present invention are combinatorial fluorophore array (CFA) libraries which comprise multiple solid supports or multiple locations on a solid support, each support or location having attached thereto an oligomer comprising the subject fluorescent nucleoside analogs. A combinatorial fluorophore array (CFA) library may also comprise one or more unlabeled nucleosides wherein the one or more unlabeled nucleosides are positioned 5xe2x80x2 or 3xe2x80x2 to the oligomer of fluorescent nucleoside analogs or interspaced between the fluorescent nucleoside analogs. In addition, a CFA library may further comprise one or more non-fluorescent nucleotide analogs selected from the group consisting of cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside wherein said one or more non-fluorescent nucleotide analogs is interspaced between the flourescent nucleotide analogs or between the fluorescent nucleoside analogs and the and non-labeled nucleosides.
The present invention also provides a method of selecting a fluorophore suitable for use in labeling a nucleic acid molecule which comprises constructing a subject combinatorial fluorophore array library and selecting a fluorophore emitting the most intense fluorescence or emitting a specific wavelength of light.
Also provided is a method of identifying a fluorophore emitting a large Stokes shifts which comprises constructing a subject combinatorial fluorophore array library, exciting the library at short wavelength, and selecting a fluorophore which emits light at a much longer wavelength.
A method of identifying a fluorophore involved in energy transfer is also provided. The method comprises constructing a subject combinatorial fluorophore array library, hybridizing a nucleic acid comprising a donor or acceptor dye to a nucleic acid sequence in the CFA library and correlating any change in color exhibited by the hybridized molecules with energy transfer. Members of the library which give greater changes in acceptor emission intensity are most efficient at energy transfer.
A method for identifying a fluorophore sequence that changes its emission wavelength or intensity on binding an analyte is also provided. The method comprises constructing a subject combinatorial fluorophore array library, incorporating an analyte affinity molecule, allowing an analyte solution to contact the library, and selecting library members that change emission intensity or wavelength on binding of the analyte molecule.
The present invention also provides oligonucleotide analogs comprising one or more of the subject nucleoside analogs in place of a DNA or RNA base. Further, the subject oligonucleotide analogs may comprise a modification to the sugar-phosphate backbone such as that found in phosphorothioate DNA, 2xe2x80x2-O-methyl RNA, phosphoramidite DNA, 2xe2x80x2fluoro DNA, peptide nucleic acid (PNA) or alpha DNA.